Abstract:Objective:To investigate effects of activated G-protein coupled estrogen receptor(GPER) signaling pathway induced by estrogen on the production of interleukin-6 (IL-6) in ER-negative breast cancer cells. Methods:After treatment of SKBR-3 and MDA-MB-453 cells,the expression of IL-6 mRNA was measured by Real-time qPCR. The secretion of IL-6 in supernatant was detected by ELISA. The protein expression level of p-ERK and p-AKT was determined by Western blot. Results:17-β estradiol (E2) and GPER specific agonist (G1) significantly increased the mRNA expression of IL-6 in SKBR-3 and MDA-MB-453 cells,which could be blocked by GPER specific antagonist (G15). After treatment with E2 and G1,GPER/ERK and GPER/AKT signaling pathways were remarkably activated to promote the protein expression of p-ERK and p-AKT (P < 0.05). The relative protein expressions of p-AKT and p-ERK in the E2 and G1 treatment groups were (4.16 ± 0.65),(3.21 ± 0.45) and (2.87 ± 0.42),(2.64 ± 0.24) times than those of the control group,respectively,and the same results were obtained in MDA-MB-453 cells. Interestingly,these changes induced by E2 and G1 were significantly blocked by the MEK inhibitor U0126 rather than PI3K inhibitor Wortmannin (P < 0.05). Conclusion:Estrogen enhances the expression and secretion of IL-6 in ER(-) breast cancer cells,which may associates with the up-regulation of GPER/ERK signaling pathway,and the inflammatory microenvironment mediated by GPER may play an important role in the development of ER(-) breast cancer.

Abstract:Objective:To observe the effect of endothelial protein C receptor(EPCR)on proliferation,migration and angiogenesis of human umbilical vein endothelial HUVECs cells,and to explore the role of EPCR in tumor angiogenesis. Methods:siRNA was performed to silence the expression of EPCR in human breast cancer cell line MDA-MB-231. The expression of EPCR in siRNA transfection was identified by reverse tansciption polymerse chain reaction(RT-PCR)and Western Blot. The experiment included three groups:the EPCR siRNA group,the irrelevant sequence group and the control group. HUVEC cells were cultured under tumor microenvironment,which was simulated by tumor conditioned medium. CCK-8 kit was performed to detect the endothelial cells proliferation,the transwell method was for detection of endothelial cell migration numbers,and matrigel in vitro small tube formation assay was performed to survey the state of tube formation. Results:The EPCR siRNA group showed lower cell proliferation activity,less number of cell migrats and tube formation than the other two groups (P﹤0.05). Conclusion:Low-expressed EPCR in human breast cancer cell line MDA-MB-231 can inhibit the proliferation,migration and angiogenesis of endothelial cells,which shows that EPCR may play an important role in tumor angiogenesis.

Abstract:Objective:To investigate effects of human anti-Trop-2 Fab antibody on biological characteristics of cervical cancer cells. Methods:The expression of Trop-2 protein from cervical cancer cells was assessed by flow cytometry(FCM)and immunofluores- cence. The effect of human anti-Trop-2 Fab antibody on migration in human cervical cells was examined by wound healing assay and transwell test. Cell apoptosis induced by Trop-2 Fab was analyzed by flow cytometry. Human anti-Trop-2 Fab antibody influence on cervical cell proliferation was detected by cell counting kit-8 (CCK8)assay. Results:Trop-2 protein was confirmed to express on human cervical cells. Human anti-Trop-2 Fab antibody inhibited human cervical cancer cell growth,migration and invasion,at the same time induced cervical cancer cell apoptosis. Conclusion:Human anti-Trop-2 Fab antibody can significantly inhibit the malignant biological behavior of cervical cancer cells. This fusion antibody may play a role in tumor targeting therapy,and Trop-2 may be a potential therapeutic target for cervical cancer.

Abstract:Objective:To study the radioresistance of esophageal cancer stem cells. Methods:The ECA109 cells were cultured in serum-free medium to enrich esophageal stem-like cells. MTT assay was performed to detect the multiplication capacity of cancer cells treated with different concentrations of cyclooxygenase-2 (COX-2) selective inhibitor NS398 (2.5,5.0,10.0,20.0,40.0 and 80.0 μmol/L). Sensitization effects of parental cells and spheroid cells were evaluated by clone formation assay. Sphere-forming assay was aimed to analyze the effect of NS398 combined with X-ray radiation on cell spheres formation. Western blot was performed to determine the expression level of β-catenin. Results:NS398 inhibited the proliferations of parental cells and spheroid cells in a time-and concentration-dependent way. After treated with 20 μmol/LNS398,Do,Dq and SF2 values of parental cells were all decreased (P < 0.05),and the sensitization enhancement ratio (SER) was 1.17. Do and SF2 values of spheroid cells were decreased (P < 0.05),and the SER was 1.12. Formation rate of the cell spheres was increased after irradiation (P < 0.05). Compared with the radiation alone group,the formation rate of the cell spheres and β-catenin expression of parental cells and spheroid cells were decreased in the combination group (t = 7.01,P < 0.01;t = 10.15,P < 0.01;t = 3.225,P < 0.05). Conclusion:The sensitization effect of spheroid cells is less than parental cells,which indicates that spheroid cells has the radioresistance. It may be correlated with the inhibition of the expression level of β-catenin.

Abstract:Objective:To acquire the purified recombinant lethal factor 253 (LF253)antigen which owned biological activity and test its competitive capacity binding protective antigen (PA)compared with lethal facfor (LF)protein. Methods:The LF253 gene was amplified by PCR,and the truncated LF gene was inserted into pET-28a (+),and transferred into E.coli.BL21 (DE3) as the host strain. LF253 was expressed as a recombinant protein induced with isopropyl-β-d-thiogalactoside(IPTG). The protein was purified with His label affinity chromatography and was subjected to antigenicity by Western blot and ELISA. The affinity was detected by Biacore T-100,and the biological activity was detected by cellular toxicity test. Results:We successfully established prokaryotic expression vector pET-28a/LF253,and sLF253 was expressed and purified. Western blot and ELISA results showed that sLF253 had an excellent antigenicity. Cellular toxicity detection showed that recombinant protein neutralized the biological effects caused by lethal toxin in vitro and in vivo. Conclusion:Recombinant sLF253 can recognize PA and competitively inhibit LF polymerization with PA to block its lethal effects. This protein may lay the experimental basis for the future of anthrax vaccine research and development.

Abstract:Objective:To investigate the possible mechanisms of apoptosis occurred in islet β-cell line (INS-1) which was induced by PCB1254 in vitro. Methods:After the treatment with different concentrations of PCB1254,the cell viability of INS-1 was assayed by CCK-8. After the treatment with PCB1254 (5 μg/ml),the morphological change and apoptosis situation of INS-1 were observed by inverted microscope. The apoptosis of INS-1 was further detected by flow cytometry. The expression of Caspase-3,Bim,Bcl-2,C-Fos,P-JNK,P-P38 and P-ERK were measured by Western blot. Reactive oxygen species (ROS) level was detected by dihydroethidium (DHE) fluorescence probe. The apoptosis level of INS-1 was observed under inverted microscope after ERK inhibitor PD98059 intervening the PCB1254 treated INS-1 cells. Results:With the increasing of concentration of PCB1254,the cell viability of INS-1 declined. When the concentration was higher than 5 μg/ml,there were significant differences compared with the control group (P < 0.01). INS-1 cells apoptosis, which was induced by PCB1254 (5 μg/ml), was observed through inverted microscope. The apoptosis cells turned dark and floated in the medium. Flow cytometry assay showed that PCB1254 could induce the apoptosis of INS-1 cells. Western blot showed that the expressions of apoptosis related Cleaved Caspase-3 and Bim proteins were up-regulated,while the expression of anti-apoptotic protein Bcl-2 was down-regulated. The expression of oxidative stress related protein C-Fos was up-regulated and the ROS fluorescence was enhanced. The expression of p-ERK in MAPK signal pathway was up-regulated. No obvious change of apoptosis was found under the intervention of ERK inhibitor PD98059. Conclusion:PCB1254 may induce the apoptosis of INS-1 cells through oxidative stress signaling pathway,and this process may be accompanied with the activation of ERK signaling pathway.

Abstract:Objective:To investigate whether systematic implantation of bone marrow mesenchymal stem cells(BMMSCs)can promote the survival of expanded flap. Methods:BMMSCs of rats were isolated through the whole bone marrow adherent method and propagated to the third generation. When 90% cells were confluent,BMMSCs were digested and cultured. After three passages,cell immunophenotype of CD29+,CD90+,CD34- and CD45- were determined. A total of 20 Sprague-Dawley (SD)rats,weighing 300-350 g,were randomly divided into 2 groups equally:the control group and the treatment group. In the control group,a dorsal expanded skin flap across 3 areas was harvested and sutured back. In the treatment group,1 h after the harvest and suturing back of a dorsal expanded flap,1 ml of suspension of BMSC/100 g(1×106 cell/ml) was injected in the rats through the tail veins. One week later,the necrotic flaps of rats in both groups and the diameter of choke vessels between the iliolumbar angiosome and the intercostal angiosome were measured and compared. Results:Positive expressions of CD29,CD9,CD34 and CD45 were 99.3%,93.5%,0.82% and 2.22%,respectively,in the third generation of cell culture. One week after flap harvest,the necrotic rate of the flap was (29.4 ± 4.2)% in the control group,which was significantly larger than that in the treatment group[(10.4 ± 3.3)%,P < 0.001]. The diameter of choke vessels between the iliolumbar angiosome and the intercostal angiosome in the control group was(183 ± 25)μm,which was statistically smaller than that in the treatment group[(226 ± 23)μm,P = 0.001]. Conclusion:BMMSCs can obviously promote survival of expanded flap in rats through enhancing the dilation of choke vessels,and the mechanism of which remains to be further investigated.

Abstract:Objective:To investigate the role of plasminogen activator inhibitor-1 (PAI-1) in the development and spontaneous regression of liver fibrosis. Methods:Rat liver fibrosis models were induced by subcutaneous injection of carbon tetrachloride(CCl4) and tissue samples were obtained for study at various times. The different stages of fibrosis were confirmed with HE and VG staining,and then the expression of PAI-1 in these tissue samples was detected by reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry. Results:There was only weak expression of PAI-1 detected in endochylema located in portal area of normal liver,and the expression of PAI-1 was mainly located within the area of hepatic sinusoid and endochylema in fibrotic liver. The PAI-1 expression increased accordingly with the development and progression of fibrosis while decreased in spontaneous regression. Semiquantitative analysis showed that the expression of PAI-1 for normal control and 2,4,6 weeks after CCl4 administration were 0.142 ± 0.030,0.361 ± 0.048,0.757 ± 0.068 and 0.838 ± 0.048,the expression of PAI-1 for 2,4,6 weeks after resolution of fibrosis were 0.613 ± 0.054,0.524 ± 0.060 and 0.210 ± 0.044. Semiquantitative analysis by RT-PCR showed that the mRNA expression of PAI-1 in the model group treated with CCl4 for 2,6,8 weeks was higher than that in normal control,increasing to 6.83 ± 2.60,12.43 ± 2.65 and 26.32 ± 5.17 fold. Nevertheless,the mRNA expression of PAI-1 decreased with the resolution of fibrosis after stopping CCl4 induction. The mRNA expression of PAI-1 for 2,4,6 weeks after resolution of fibrosis was 17.86 ± 4.6,14.62 ± 5.99 and 11.21 ± 1.98 times,respectively,compared with normal control. Conclusion:PAI-1 was up-regulated in liver fibrosis development while down-regulated in spontaneous regression,which indicated that it may play an important role in the development and progression of hepatic fibrosis.

Abstract:Objective:To observe the expression of interleukin (IL)-17 in asthmatic mice and to investigate the effect of IL-17 on release of IL-6 in mast cells. Methods:BALB/c mice were randomly divided into control group and asthma group,and treated with equal amount of phosphate buffer solution (PBS)and ovalbumin (OVA),respectively. Mouse asthma model was validated by detecting the total number of cells and eosinophils (EOS)in bronchoalveolar lavage fluid (BALF). The expression differences between IL-17 mRNA in lung tissues and IL-17 in BALF and serum were measured by reverse transcription-polymerase chain reaction(RT-PCR)and enzyme-linked immunosorbent assay (ELISA)in the two groups. Mouse P815 mast cells were divided into the control group and the IL-17 group,and treated with equal amount of culture medium and IL-17,respectively. The levels of released IL-6 in supernatants were detected by ELISA. Results:In BALF,the total number of cells and EOS in the asthma group were significantly increased than those in the control group(P < 0.05),and the model was validated. The expressions of IL-17 mRNA in lung tissues and IL-17 in BALF and serum were significantly increased in the asthma group than those in the control group(P < 0.05). In P815 cell supernatants,the secretion of IL-6 in the IL-17 group was significantly increased than that in the control group(P < 0.05). Conclusion:The expression of IL-17 was increased in OVA-induced mouse asthma model. IL-17 may promote the process of asthma by stimulating IL-6 release by mast cells.

Abstract:Objective:To investigate the influence of interleukin-18 (IL-18) on the rat deep vein thrombosis (DVT). Methods:Viral vectors of IL18-pCDH-GFP/IL18-LMP-shRNAmir1 were constructed. SD rats (n=27) were randomly divided into IL-18 overexpression group,IL-18 inhibition group and control group which were injected with 100 μl IL-18 overexpression lentivectors (IL18- pCDH-GFP),100 μl IL-18 inhibition retroviral vectors (IL18-LMP-shRNAmir1),100 μl saline,respectively,into the tail vein. All rats’ inferior vena cavas (IVC) were modeled as “stenosis” to promote IVC thrombosis after 24 h of injection. The weight and length of IVC thrombosis after 24 h of modeled were investigated,and the expression of IL-18 in the venous wall was measured by real-time PCR assay. Results:IL18-pCDH-GFP and IL18-LMP-shRNAmir1 vectors showed the ideal overexpression/inhibition rate in vitro. All groups had stabilized thrombus formation after modeled 24 h. The average length and weight of thrombus in the IL-18 overexpression group were significantly higher than those of other groups (P < 0.05). The level of IL-18 in the overexpression group was remarkably higher than that of other groups in the vessel wall (F=3.784,P < 0.05),which was proved by real-time PCR assay. Conclusion:IL-18 promotes thrombus formation in rats,and inhibition of IL-18 reduces the thrombus formation. IL-18 may be related to the development process of DVT and its proinflammatory effect may play a vital role in the pathogenesis of VTE.

Abstract:Objective:We sought to determine whether single nucleotide polymorphisms(SNPs) in toll-like receptor(TLR) 2 subfamily genes affect the risk of asthma in adults from southeastern China,and the association between SNPs and asthma clinical phenotypes. Methods:A total of 318 asthmatic patients and 352 non-asthmatic controls were recruited. Eight SNPs in TLR2 subfamily genes were detected using Genome Lab SNP stream. Each SNP and haploid type as well as asthma phenotype were analyzed. Results:We found that patients with the TLR2/rs7656411 TT variant homozygote had a significantly reduced risk of asthma when compared with those with the GG wild-type homozygote[adjusted odds ratio (OR):0.63; 95% confidence interval (CI):0.41-0.98; P = 0.41]. There was no significant difference between rs7656411TT+GT and asthma (OR:0.77; 95% CI:0.54-1.09; P≥ 0.05). Furthermore,a positive association was observed between the T allele of rs2381289 in TLR6 and allergic rhinitis in asthma(OR:1.79; 95% CI:1.10-2.91; P = 0.025),while the A allele of rs11466651 in TLR10 was negatively associated with allergic rhinitis(OR:0.49; 95%CI:0.26-0.95; P = 0.046). Conclusion:Our results indicate that a genetic variant in the TLR2 subfamily may play a role in susceptibility to asthma.

Abstract:Objective:To investigate whether I198T and A379V polymorphisms in lipoprotein-associated phospholipase A2(Lp-PLA2)correlate with severity and stability of coronary atherosclerosis. Methods:V279F and A379V polymorphisms in Lp-PLA2 gene of 268 patients with coronary atherosclerosis and 113 controls without coronary atherosclerosis were genotyped by DNA sequencing instrument and analyzed by statistical methods. Results:Stratified by Gensini score and clinical types,no significant associations were observed between subgroups and the controls in I198T and V279F genotypes and allele frequencies. As for subgroups stratified by numbers of diseased coronary branches,only subjects with one diseased coronary branches carried higher frequencies of genotype IT+TT,VF+FF and F allele as compared to the controls. No associations were observed between patients with multi-vessel lesions and controls in I198T and V279F. In the further analysis of risk factors,blood fat levels and proportions of diabetes and hypertension patients in subjects carrying IT+TT and VF+VV genotypes showed no difference with II and VV genotypes. Conclusion:No associations existed between I198T and V279F polymorphisms in Lp-PLA2 and the severity and stability of coronary atherosclerosis.

Abstract:Objective:To investigate the diagnostic value of serum and hepatic tissue Golgi protein 73 (GP73) and phosphatidy- linositol proteinglycan 3 (GPC3) in hepatocellular carcinoma (HCC). Methods:The peripheral serum and tissue samples from 70 individuals were collected,including 39 HCC patients and 31 cirrhotic patients. The expression levels of serum GP73 and GPC3 were detected by ELISA;the expression levels of tissue GPC3 mRNA and GP73 mRNA were measured by RT-PCR. The differences of GP73 and GPC3 expression between liver cancer group and cirrhosis group were observed. The correlation between GP73 and GPC3 expression and clinical stage of HCC was analyzed,and the correlation between GP73 and GPC3 expression and pathological grade of HCC was also analyzed. Results:The expression level of serum GP73 and GPC3 from the HCC patients was significantly higher than those from the cirrhotic group (P < 0.001). The GPC3 value for the cutting off was 9.3 μg/L,the sensitivity was 89.74%,the specificity was 96.77%,the positive predictive value was 97.20%,and the negative predictive value was 88.20%. The GP73 value for the cutting off was 77.68 ng/ml,the sensitivity was 92.31%,the specificity was 83.87%,the positive predictive value was 87.80%,and the negative predictive value was 89.70%. The expression levels of GP73 and GPC3 in HCC patients show no statistic difference between age,gender,tumor size and clinical stage;nevertheless,the expression level of GPC3 in HCC patients seemed to be correlated with tumor pathological grade. Conclusion:Over expression of liver GPC3 and GP73 were associated with HCC,and quantitative detection of serum GPC3 and GP73,which were more sensitive than AFP,should be a useful marker for diagnosis of HCC.

Abstract:Objective:To explore the application value of ultrasound elastic strain ratio in diagnosing thyroid nodules of TI-RADS 4-6 categories. Methods:Conventional ultrasound was applied to 324 thyroid nodules (from 303 patients) to observe their two-dimensional ultrasonographic characteristics. The 324 thyroid nodules were classified into different groups according to the TI-RADS,and then undergone ultrasonic elastography and the strain ratios of them were recorded. A receiver-operating characteristic curve(ROC)was used to identify the cut-off point in the differential diagnosis of thyroid nodules. The 137 thyroid nodules of TI-RADS 4-6 categories were classified into three groups to analyze the application value of strain ratio in each group:group Ⅰ,TI-RADS 4A category;group Ⅱ,TI-RADS 4B category;group Ⅲ,TI-RADS 5-6 categories (the pathologically confirmed cases in category 6 have been removed). Results:The best boundary value of strain ratio was 0.55. The difference of strain ratio in group I was not statistically significant,while those in group Ⅱ and group Ⅲ were statistically significant. The sensitivity,specificity,accuracy and positive predictive value in group Ⅱand group Ⅲ were 90.9%,75.0%,87.5%,93.0% and 85.7%,80.0%,84.6%,94.7%,respectively. Conclusion:The combination of TI-RADS classification system and strain ratio in group Ⅱ and group Ⅲ was of high application value,which suggests that the malignant nodules should be given early surgical treatment in stead of examination like fine needle aspiration biopsy. While in group I,the application value of strain ratio was limited,thus other examination methods should be used at the same time as early as possible to avoid delays in the diagnosis of nodules.

Abstract:Objective:To investigate the status of BMP/Smads signaling pathway on the bone of lupus mice(MRL/lpr). Methods:The femurs of the mice were isolated,and the bone structure was detected by hematoxylin-eosn(HE)staining. The protein expression levels of bone morphogenetic protein-2 (BMP-2)were measured by immunohistochemistry. Bone marrow mesenchymal stem cells(BMMSCs)were isolated by the density gradient centrifugation and the adherence screening methods. After BMMSCs growing on coverslips,the protein of BMP-2 and PSmad1/5/8 was valued by immumofluorescence method. The mRNA expressions of ALP,Runx2 of BMMSCs were measured by Real-time PCR,and the Alkaline phosphatase (ALP)staining was performed for measuring the early osteogenic differentiation induced by BMP-2. Results:Compared with the controlled mice,the cortex of MRL/lpr mice were reduced (P < 0.01). There was no difference in the BMP-2 expression on the bone of the two groups by immunohistochemistry detection(P > 0.05). The expressions of BMP-2 and PSmad1/5/8 in BMMSCs of lupus mice were lower than those of the control group(P < 0.05). After 7 days of BMP-2 stimulation,ALP activities of BMMSCs from lupus mice were decreased compared with the control group. The mRNA levels of ALP in lupus mice were lower than that of C3He/HeJ mice (P < 0.01),while there were no differences in the Runx2 mRNA levels between the two groups after BMP-2 stimulation(P > 0.05). Conclusion:BMP/Smads signaling was inhibited in the bone of lupus mouse.

Abstract:Objective:To investigate relationship between DNA polymerase iota (Polι) and lymph node metastasis of human esophageal squamous cell carcinoma. Methods:Polι expression in esophageal squamous cell carcinoma tissues was detected by Real-time PCR. Mann-Whitney U test was performed to analyze the relationship between Polι and lymph node metastasis. Polι and Nm23 expression in esophageal squamous cell carcinoma tissues was detected by immumohistochemical staining. Spearman correlation analysis was performed to analyze the relationship between Polι and Nm23. The Polι expression vector was transfected into esophageal cancer cells TE-1 and ECA-109 to up-regulate Polι expression. Polι and Nm23 mRNA expression was detected by Realtime-PCR. Transwell chamber assay was performed to analyze cell invasional ability. Results:In clinical tissue samples,Polι expression was positively correlated with the lymph node metastasis of human esophageal squamous cell carcinoma (P < 0.01),and negatively correlated with Nm23 in protein level (R=-0.481,P < 0.05). In vitro study,overexpression of Polι enhanced cell invasion (P < 0.05) and significantly inhibited Nm23 mRNA expression in TE-1 and ECA-109 cells (P < 0.001). Conclusion:Polι is closely related with the invasion and metastasis of esophageal squamous cell carcinoma,the possible mechanism of the regulation is through down regulating the expression of Nm23.

Abstract:Objective:To analyze the CD4+T variation and its impact factors on the HIV/AIDS,who initially had received highly active antiretroviral therapy (HAART) for 1 year in Jiangsu province. Methods:According to the baseline and follow-up data of HIV/AIDS initially receiving HAART with the CD4+T cell count tested in baseline and 1 year after treatment,the deadline of the follow-up was on May 31,2014. Excel database was established and statistical analysis was performed using SPSS 16.0 software. Results:There were 3 290 patients initially receiving HAART with the CD4+ T cell count tested in baseline and 1 year after treatment totally. Eighty-one point four percent of them were from local province; the ratio of male to female was 4.36:1; the average age was 39.7 ± 12.1 years old; 92.3% of the patients were infected with HIV through sexual transmission; the mean CD4+ T cell count of cases was 185.81 cells/μl in baseline,and it increased to 312.20 cells/μl 1 year after treatment and significant differences were found. The age,CD4+T baseline,WHO disease stage,treatment site and regimen influenced the growth value of CD4+ T. Conclusion:HIV/AIDS treatment has a remarkable effect on immunological function rehabilitate for pationts in Jiangsu province. Standardized and early treatment still should be strengthened.